A preparation method of composite water purification bacterial agent capsule

Abstract

The invention discloses a preparation method of a composite water purification bacterial agent capsule, which specifically comprises the following steps: mixing Fe(NO3)3, attapulgite and water and then standing to prepare Fe(NO3)3. 3+ Modified attapulgite; Fe 3+ Modified attapulgite is mixed with unmodified attapulgite, water is added and stirred to prepare attapulgite floc suspension; concentrated bacterial liquid of Bacillus pumilus and Bacillus cereus is cultivated; floc suspension of Bacillus pumilus and Bacillus cereus and attapulgite is prepared; the water content of the floc suspension is controlled to 40% to 50% to obtain mud, sodium alginate solution is added to the mud, and plastic mud is obtained after stirring; the plastic mud is prepared into spherical particles in a mold, and the bacterial agent is embedded and freeze-dried to obtain a finished composite water purification bacterial agent capsule. The composite water purification bacterial agent capsule prepared by the method of the present invention can quickly settle in the water body, and can continuously release high-efficiency water purification bacteria to the water body at the settlement location, improve the microecological bacterial phase structure of the water body, and exert a long-term water purification effect.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial agent immobilization embedding, relates to the preparation of water purification bacterial agent, and particularly to a preparation method of a composite water purification bacterial agent capsule. Background Art

[0002] With the development of social economy and the expansion of urban population, the discharge of various industrial wastewater and domestic sewage has increased significantly. However, the sewage collection and treatment capacity of most cities is relatively backward, and a large amount of sewage is often discharged into urban rivers without treatment, resulting in serious pollution of rivers running through towns and even seasonal and perennial black and smelly water.

[0003] Water pollution control is a systematic project. It is necessary to control pollution at the source by relying on various source control and pollution interception projects, and to adopt a variety of means to control and ecologically restore existing polluted water bodies. Microbial water purification agents have significant advantages in the restoration of polluted water bodies, such as low cost, simple construction, quick results and no negative environmental impact. However, most of the water purification agents on the market are prepared by adding adsorbents and excipients to bacterial fermentation broth, and then drying and forming. Such water purification agents usually have shortcomings such as short shelf life and difficulty in evenly dispersing microorganisms in water bodies. There are also microcapsule agents prepared by embedding methods. This type of preparation method can significantly improve the shelf life and the tolerance of strains to the water environment, but most of the artificially added agents are lost in the river with the flow of water, and cannot continuously exert the water purification effect. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for preparing a composite water-purifying bacterial agent capsule. The composite water-purifying bacterial agent capsule prepared by the method of the present invention can quickly settle in a water body, and can continuously release highly efficient water-purifying bacteria into the water body at the settling point, thereby improving the microecological bacterial phase structure of the water body and exerting a long-term water purification effect.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing a composite water purification bacterial agent capsule comprises the following steps:

[0007] Step (1), Fe(NO 3 ) 3 , attapulgite and water are mixed and stirred and then allowed to stand. After filtering, the filter cake is dried and then roasted, and then ground and sieved to obtain Fe 3+ Modified attapulgite;

[0008] Step (2), the Fe obtained in step (1) 3+ The modified attapulgite and the unmodified attapulgite are mixed, water is added and stirred to prepare an attapulgite floc suspension;

[0009] Step (3), inoculating Bacillus pumilus and Bacillus cereus into liquid culture medium for enrichment culture respectively, discarding the supernatant after centrifugation, collecting the bacterial sludge precipitate at the lower layer, adjusting the concentration of the bacterial sludge precipitate with physiological saline, and preparing a concentrated bacterial solution;

[0010] Step (4), adding the concentrated bacterial solution of Bacillus pumilus and the concentrated bacterial solution of Bacillus cereus obtained in step (3) to the attapulgite floc suspension obtained in step (2), and oscillating and stirring to allow the strains to be stably fixed on the surface and gaps of the attapulgite flocs;

[0011] Step (5), controlling the water content of the mixed solution obtained in step (4) to 40% to 50% to obtain mud, adding sodium alginate solution to the mud, and stirring to obtain plastic mud;

[0012] Step (6), preparing the plastic mud obtained in step (5) into spherical particles in a mold, and immediately placing the particles in a calcium chloride solution, stirring to complete the embedding of the bacterial agent, separating the embedded spherical particles, washing the residual calcium chloride with clean water, and freeze-drying the particles to obtain a finished composite water purification bacterial agent capsule.

[0013] A further improvement of the present invention is:

[0014] In step (1), Fe(NO 3 ) 3 The mass ratio of attapulgite and water is 0.8~1.2:0.8~1.2:100, the standing time is 10~14h, the drying temperature is 100~110℃, the roasting temperature is 350~450℃, the roasting time is 2~4h, and it is ground through a 200-mesh sieve.

[0015] Furthermore, in step (2), Fe 3+ The mass ratio of modified attapulgite to unmodified attapulgite is 1.8~2.2:1, and the mass fraction of solid components in the attapulgite floc suspension is 8~12%.

[0016] Furthermore, the liquid culture medium in step (3) includes 5 g beef extract, 10 g peptone, 5 g sodium chloride, and 1000 mL sterile water.

[0017] Furthermore, the culturing process in step (3) is as follows: culturing in a shaking incubator at 35-40°C for 22-26 hours; the centrifugation temperature is 2-6°C, the speed is 3500r / min-4500r / min, and the time is 10-15min; the mass percentage concentration of the physiological saline is 0.5-1.5%, and the concentration of the concentrated bacterial solution is 10 10 CFU / mL.

[0018] Furthermore, in step (4), the volume ratio of the concentrated Bacillus pumilus solution to the concentrated Bacillus cereus solution is 1:0.8-1.2, and 0.8-1.2 g of the attapulgite suspension is required to mix 1 mL of the mixed solution of the concentrated Bacillus pumilus solution and the concentrated Bacillus cereus solution, and the oscillating stirring temperature is 35-40° C. and the time is 22-26 h.

[0019] Furthermore, in step (5), the water content of the mixed solution is controlled by vacuum filtration, the mass of the sodium alginate solution is 3% to 5% of the mass of the mud, and the concentration of the sodium alginate solution is 20 to 40 mg / ml.

[0020] Furthermore, in step (6), spherical particles with a particle size of 2 to 5 mm are prepared in a mold, the concentration of the calcium chloride solution is 15 to 25 mg / ml, and the freeze-drying process is: first pre-freeze in a -22 to -18°C refrigerator for 1.5 to 2.5 hours, and then place in a -40 to -35°C, 5.5 to 6.5 Pa vacuum freezer for 22 to 26 hours.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] First, the Bacillus pumilus and Bacillus cereus used in the present invention are facultative oligotrophic water purification strains. After being embedded, the strains can play a water purification role under the concentration gradient of individual pollutants and can be used for purifying water bodies with different pollution degrees.

[0023] Second, the present invention adopts Fe-modified attapulgite and original attapulgite to form a floc structure with a positive charge on the surface, which makes it easier for microorganisms to attach and form on the carrier surface; the floc structure has abundant void space, which can greatly increase the bacterial load; the modified attapulgite has a large density, and when used as a carrier, the bacterial agent has sufficient density to ensure rapid sinking after being placed, providing a guarantee for the fixed-point planting of excellent water purification strains.

[0024] Third, the present invention uses sodium alginate and calcium chloride to cross-link to form a wall material to embed the bacterial agent, and the strain can be continuously and slowly released in the water body; after the capsule wall material is degraded, the embedded bacterial agent can continue to form a biofilm with attapulgite as a carrier and continuously released into the water body.

[0025] Fourthly, the concave soil used in the present invention is an excellent adsorbent, and has a strong adsorption capacity for organic matter / nitrogen, phosphorus and other nutrients in the water body, can achieve nutrient enrichment, and can provide continuous support for the growth and reproduction of microorganisms, thereby achieving microbial colonization. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is an appearance diagram of the composite water purifying bacteria capsule prepared by the present invention.

[0027] Figure 1 The finished capsules are light yellow spherical particles. DETAILED DESCRIPTION

[0028] The following is a detailed description of the embodiments of the present invention.

[0029] Detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0030] In this application, Bacillus pumilus and Bacillus cereus used are two highly efficient facultative water-purifying oligotrophic bacteria purified and isolated by the inventor's research group. The separation process can be found in the document "Research on Screening Oligotrophic Water-Purifying Microorganisms by Gradient Nutrient Dilution Method" (Hubei Agricultural Sciences, 2022, Vol. 61: 50-53.). Example 1

[0031] (1) Modified attapulgite: Fe(NO 3 ) 3 , attapulgite and water are mixed in a mass ratio of 1:1:100, stirred and allowed to stand for 12 hours, filtered and dried at 105°C, calcined at about 400°C for 3 hours, and then ground through a 200-mesh sieve to obtain modified attapulgite.

[0032] (2) Preparing a suspension of attapulgite flocs: The modified attapulgite, unmodified attapulgite and water are mixed and stirred in a mass ratio of 2:1:27 to obtain a suspension of attapulgite flocs.

[0033] (3) Preparation of concentrated bacterial solution: Inoculate Bacillus pumilus and Bacillus cereus into liquid culture medium respectively, and culture in a shaking incubator at 37°C for 24 hours. Centrifuge at 4°C and 4000r / min for 12 minutes, save the lower layer of concentrated bacterial solution, and adjust the concentration of the concentrated bacterial solution to 10 with 0.9% saline. 10 CFU / mL.

[0034] (4) The concentrated bacterial solutions of Bacillus pumilus and Bacillus cereus were mixed in a volume ratio of 1:1, and then added to the modified attapulgite floc solution at a mass ratio of 1 mL (mixed concentrated bacterial solution) / g (attapulgite). The mixture was stirred and shaken at 37 °C for 24 h to allow the strains to stably adhere to the surface of the attapulgite flocs and the voids of the flocs, thereby obtaining a mixed solution of attapulgite and the strains.

[0035] (5) Preparation of plastic mud: The mixed solution is filtered under reduced pressure to obtain plastic mud with a water content of 40%, and 30 mg / ml sodium alginate solution with a mass ratio of 5% is added, and the mixture is stirred evenly to obtain plastic mud.

[0036] (6) Preparation of composite water purification bacterial capsules: The plastic clay was placed in a mold and pressed into spherical particles with a particle size of 3 mm, and immediately demolded and placed in a 20 mg / ml calcium chloride solution and stirred for about 30 min. After the embedded spherical particles were separated, the residual calcium chloride was washed with clean water, and the particles were pre-frozen in a -20°C refrigerator for 2 h, and then placed in a -40°C, 5.9 Pa vacuum freezer for 24 h to obtain freeze-dried composite water purification bacterial capsules. Example 2

[0037] (1) Modified attapulgite: Fe(NO 3 ) 3 , attapulgite and water are mixed in a mass ratio of 0.8:0.8:100, stirred and allowed to stand for 12 hours, filtered and dried at 100°C, calcined at about 350°C for 3 hours, and then ground through a 200-mesh sieve to obtain modified attapulgite.

[0038] (2) Preparing a suspension of attapulgite flocs: The modified attapulgite, unmodified attapulgite and water are mixed and stirred in a mass ratio of 1.8:1:25 to obtain a suspension of attapulgite flocs.

[0039] (3) Preparation of concentrated bacterial solution: Inoculate Bacillus pumilus and Bacillus cereus into liquid culture medium respectively, and culture in a shaking incubator at 35°C for 22 hours. Centrifuge at 2°C and 3500r / min for 12 minutes, keep the lower layer of concentrated bacterial solution, and adjust the concentration of the concentrated bacterial solution to 10 with 0.9% saline. 10 CFU / mL.

[0040] (4) The concentrated bacterial solutions of Bacillus pumilus and Bacillus cereus were mixed at a volume ratio of 1:0.8, and then added to the modified attapulgite floc solution at a mass ratio of 0.8 mL (mixed concentrated bacterial solution) / g (attapulgite). The mixture was stirred and shaken at 35 °C for 22 h to allow the strains to stably adhere to the surface of the attapulgite flocs and the voids of the flocs, thereby obtaining a mixed solution of attapulgite and the strains.

[0041] (5) Preparation of plastic mud: The mixed solution is filtered under reduced pressure to obtain plastic mud with a water content of 40%, and a 30 mg / ml sodium alginate solution with a mass ratio of 3% is added, and the mixture is stirred evenly to obtain plastic mud.

[0042] (6) Preparation of composite water purification bacterial capsules: The plastic clay was placed in a mold and pressed into spherical particles with a particle size of 2 mm, and immediately demolded and placed in a 15 mg / ml calcium chloride solution and stirred for about 30 min. After the embedded spherical particles were separated, the residual calcium chloride was washed with clean water, and the particles were placed in a -22°C refrigerator for pre-freezing for 1.5 h, and then placed in a -40°C, 5.5 Pa vacuum freezer for 22 h to obtain freeze-dried composite water purification bacterial capsules. Example 3

[0043] (1) Modified attapulgite: Fe(NO 3 ) 3 , attapulgite and water are mixed in a mass ratio of 1.2:1.2:100, stirred and allowed to stand for 14 hours, filtered and dried at 110°C, calcined at about 450°C for 4 hours, and then ground through a 200-mesh sieve to obtain modified attapulgite.

[0044] (2) Preparing a suspension of attapulgite flocs: The modified attapulgite, unmodified attapulgite and water are mixed and stirred in a mass ratio of 2.2:1:28 to obtain a suspension of attapulgite flocs.

[0045] (3) Preparation of concentrated bacterial solution: Inoculate Bacillus pumilus and Bacillus cereus into liquid culture medium respectively, and culture in a shaking incubator at 40°C for 22 hours. Centrifuge at 6°C and 4500r / min for 10 minutes, keep the lower layer of concentrated bacterial solution, and adjust the concentration of the concentrated bacterial solution to 10 with 0.9% saline. 10 CFU / mL.

[0046] (4) The concentrated bacterial solutions of Bacillus pumilus and Bacillus cereus were mixed at a volume ratio of 1:1.2, and then added to the modified attapulgite floc solution at a mass ratio of 1.2 mL (mixed concentrated bacterial solution) / g (attapulgite). The mixture was stirred and shaken at 37 °C for 24 h to allow the strains to stably adhere to the surface of the attapulgite flocs and the voids of the flocs, thereby obtaining a mixed solution of attapulgite and the strains.

[0047] (5) Preparation of plastic mud: The mixed solution is filtered under reduced pressure to obtain plastic mud with a water content of 50%, and 40 mg / ml sodium alginate solution with a mass ratio of 5% is added, and the mixture is stirred evenly to obtain plastic mud.

[0048] (6) Preparation of composite water purification bacterial capsules: Place the plastic clay in a mold and press it into spherical particles with a particle size of 5 mm, then immediately demold it and place it in a 20 mg / ml calcium chloride solution and stir for about 40 minutes. After the embedded spherical particles are separated, wash off the residual calcium chloride with clean water, place them in a -18°C refrigerator for pre-freezing for 2 hours, and then place them in a -35°C, 6.5 Pa vacuum freezer for 22 hours to obtain freeze-dried composite water purification bacterial capsules.

[0049] Example 4: Physical and chemical properties test

[0050] The composite water purification bacterial agent capsule prepared in Example 1 was used as the object to test its physical and chemical properties. The results are as follows:

[0051] 1. The particle size of the bacterial capsule is 3mm, the density is 1.87*103kg / m3, and the equilibrium sedimentation velocity in the water can reach 1.8m / s.

[0052] 2. Stability test of bacterial capsules: The unencapsulated and encapsulated strains were stored at a constant temperature of 57°C and a relative humidity of 60-75% for 7 days, which is equivalent to one year at room temperature, and the changes in the number of live bacteria were measured. The results showed that the unencapsulated strains had basically no live bacteria detected after 5 days. However, the number of live bacteria after encapsulation did not change significantly after 7 days.

[0053] 3. When bacterial capsules were added to the river device simulated in the laboratory, the water flow velocity ranged from 1m / s to 3m / s, and the bacterial capsules could settle rapidly in the river, and could maintain a stable position in the sandy and muddy bottom matrix; in the self-circulating flow device, the inlet COD concentration was set to 40mg / L, the flow rate was 2m3 / s, and the amount of bacterial capsules added was 50g. After continuous operation for 21 days, the effluent COD removal rate reached 70%; the spherical structure of the bacterial capsules had been destroyed, and Bacillus brevis and Bacillus cereus could be cultured in both the water and the bottom sediment, indicating that the water purification strains were successfully colonized.

Claims

1. A method for preparing a composite water purification bacterial agent capsule, characterized in that: The following steps are involved: Step (1), Fe(NO3)3, attapulgite and water are mixed and stirred and then allowed to stand. After filtering, the filter cake is first dried and then roasted, and then ground and sieved to obtain Fe 3+ Modified attapulgite; Step (2), the Fe obtained in step (1) 3+ The modified attapulgite and the unmodified attapulgite are mixed, water is added and stirred to prepare an attapulgite floc suspension; Step (3), inoculating Bacillus pumilus and Bacillus cereus into liquid culture medium for enrichment culture respectively, discarding the supernatant after centrifugation, collecting the bacterial sludge precipitate at the lower layer, adjusting the concentration of the bacterial sludge precipitate with physiological saline, and preparing a concentrated bacterial solution; Step (4), adding the concentrated bacterial solution of Bacillus pumilus and the concentrated bacterial solution of Bacillus cereus obtained in step (3) to the attapulgite floc suspension obtained in step (2), and oscillating and stirring to allow the strains to be stably fixed on the surface and gaps of the attapulgite flocs; Step (5), controlling the water content of the mixed solution obtained in step (4) to 40% to 50% to obtain mud, adding sodium alginate solution to the mud, and stirring to obtain plastic mud; Step (6), preparing the plastic mud obtained in step (5) into spherical particles in a mold, and immediately placing the particles in a calcium chloride solution, stirring to complete the embedding of the bacterial agent, separating the embedded spherical particles, washing the residual calcium chloride with clean water, and freeze-drying the particles to obtain a finished composite water purification bacterial agent capsule.

2. The method for preparing a composite water purification bacterial agent capsule according to claim 1, characterized in that: The mass ratio of Fe(NO3)3, attapulgite and water in step (1) is 0.8~1.2:0.8~1.2:100, the standing time is 10~14h, the drying temperature is 100~110°C, the roasting temperature is 350~450°C, the roasting time is 2~4h, and the product is ground through a 200-mesh sieve.

3. The method for preparing a composite water purification bacterial agent capsule according to claim 1, characterized in that: Step (2) Fe 3+ The mass ratio of modified attapulgite to unmodified attapulgite is 1.8~2.2:1, and the mass fraction of solid components in the attapulgite floc suspension is 8~12%.

4. The method for preparing a composite water purification bacterial agent capsule according to claim 1, characterized in that: The liquid culture medium in step (3) includes 5 g beef extract, 10 g peptone, 5 g sodium chloride, and 1000 mL sterile water.

5. The method for preparing a composite water purification bacterial agent capsule according to claim 1, characterized in that: The culturing process in step (3) is as follows: culturing in a shaking incubator at 35-40°C for 22-26 hours; the centrifugation temperature is 2-6°C, the speed is 3500r / min-4500r / min, and the time is 10-15min; the mass percentage concentration of the physiological saline is 0.5-1.5%, and the concentration of the concentrated bacterial solution is 10 10 CFU / mL.

6. The method for preparing a composite water purification bacterial agent capsule according to claim 1, characterized in that: The volume ratio of the concentrated bacterial solution of Bacillus pumilus to the concentrated bacterial solution of Bacillus cereus in step (4) is 1:0.8-1.2, and 0.8-1.2 g of the attapulgite suspension is required to mix 1 mL of the mixed bacterial solution of the concentrated bacterial solution of Bacillus pumilus and the concentrated bacterial solution of Bacillus cereus, and the oscillating stirring temperature is 35-40° C. and the time is 22-26 h.

7. The method for preparing a composite water purification bacterial agent capsule according to claim 1, characterized in that: In step (5), the water content of the mixed solution is controlled by vacuum filtration, the mass of the sodium alginate solution is 3% to 5% of the mass of the mud, and the concentration of the sodium alginate solution is 20 to 40 mg / ml.

8. The method for preparing a composite water purification bacterial agent capsule according to claim 1, characterized in that: In step (6), spherical particles with a particle size of 2 to 5 mm are prepared in a mold, the concentration of the calcium chloride solution is 15 to 25 mg / ml, and the freeze-drying process is: first pre-freeze in a -22 to -18°C refrigerator for 1.5 to 2.5 hours, and then place in a -40 to -35°C, 5.5 to 6.5 Pa vacuum freezer for 22 to 26 hours.

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